The invention relates to the fields of microbiology and vaccine technology, and concerns the development of vaccines capable of conferring immunity to infection by group B Streptococcus.
Streptococcus agalactiae (GBS) is the leading cause of neonatal sepsis and early onset meningitis in infants in the United States, causing over 2,000 deaths each year. Thus GBS is an important disease pathogen. GBS is also responsible for more than 50,000 cases of maternal postpartum endometritis (Baker, C., In: Infectious Diseases of the Fetus and Newborn Infant. Remington J., et al. eds. Philadelphia: W. B. Saunders, (1990), pp. 742-811). Recently, GBS has increasingly been seen to cause serious infections in nonpregnant adults, primarily among the inmmunocompromised, elderly individuals and diabetics. Invasive infection has been diagnosed in 4.4 per 100,000 nonpregnant adults, with a mortality rate of 21% (Farley; M. M., et al., N. Engl. J Med 328(25):1807-1811 (1993)). This prospective surveillance study documented an annual incidence of invasive GBS disease to be 9.2 cases per 100,000 population. The incidence of invasive GBS infections in adults is higher than the incidence of infections caused by many other important pathogens, including the meningococci. Although GBS is sensitive to antibiotics, the rapid onset of the disease in neonates and infants also leads to high morbidity (50%) and mortality (20%) (Baker, C., In: Infectious Diseases of the Fetus and Newborn Infant. Remington J., et al. eds. Philadelphia: W. B. Saunders, (1990), pp. 742-811; Michel, J. L., Infectious Disease Practice 13:1-12 (1990)). Therefore, there is a need to develop vaccines capable of conferring immunity to infection by GBS.
The pathogenic streptococci express a number of surface-associated, opsonic, and protective polysaccharides and protein antigens (Kehoe, M. A., Vaccine 9:797-806 (1991); Lachenauer, C. S. and L. C. Madoff, Infect. Immun. 64:4255-4260 (1996)). The type-specific capsular polysaccharide by itself is not very immunogenic; however, antibodies to conjugates of the capsular polysaccharides and protein antigens elicit protection in animal models of GBS infections (Kasper, D., et al., in Vaccines 94, E. Norrby (ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1994), pp. 113-117; Weis, J. H. in Current Protocols in Molecular Biology, Vol. 1, F. M., Ausubel, et al., (eds.), John Wiley and Sons, Inc., New York, (1994), pp. 6.2.1-6.2.3.; Paoletti, L. C., et al., Infect. Immun. 62:3236-3243 (1994); Wessels, M. R., et al., Infect. Immun. 61:4760-4766 (1993)).
GBS also expresses a family of protective and well-characterized protein antigens called C proteins (alpha and beta) and R protein (Rib), also known as R4 (Heden, L. et al., Eur. J. Immunol. 21:1481-1490 (1991); Jerlstrom, P. G., et al., Mol. Microbiol. 5:843-849 (1991); Larsson, C., et al., Infect. Immun. 64:3518-3523 (1996); Michel, J. L., et al., in Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci, G. M. Dunny, et al., (eds.), American Society for Microbiology, Washington, D.C., (1991), pp. 214-218; Michel, J. L., et al., Infect. Immun. 59:2023-2028 (1991); Michel, J. L., et al., Proc. Natl. Acad. Sci. USA. 89:10060-10064 (1992); St{dot over (a)}lhammar-Carlemalm, M., et al., J. Exp. Med. 177:1593-1603 (1993); Wxc3xa4stfelt, M., et al., J. Biol. Chem. 271:18892-18897 (1996)). Recently an additional C-protein, the epsilon antigen was discovered (DuBois, N. B. Genetic and phenotypic properties of the surface proteins of group B Streptococcus and the identification of a new protein, Bachelor of Arts thesis in Biology, Harvard College, (1995)
The C proteins (alpha and beta, and epsilon) are surface-associated proteins on GBS carrying immunogenic epitopes that elicit protective antibodies. Antibodies raised against partially purified C proteins in rabbits were originally shown by Lancefield et al. to provide passive protection in mice against challenge with C protein-positive strains; C protein antibodies did not protect against C protein-negative strains (Lancefield, R. C., et al., J. Exp. Med. 142(1):165-179 (1975)). Strains bearing C proteins resist phagocytosis and inhibit intracellular killing (Payne, N. R, et al., J. Infec. Dis. 151:672-681 (1985)).
The C proteins have been divided into two species (Russel-Jones et al., J. Exp. Med. 160:1476-1484, 1984) that are independently expressed and antigenically distinct, and have been defined biochemically and immunologically. The alpha antigen is trypsin-resistant and has been shown to increase resistance to opsonophagocytic killing (Madoff et al., Infect. Immun. 59:2638-2644, 1991)), whereas the beta antigen is trypsin-sensitive, and binds preferentially to human serum IgA (Russel-Jones, G. J., J. Exp. Med. 160:1467-1475 (1984)) by a non-immune mechanism. Additionally, there is the epsilon antigen which is believed to be a member of the alpha antigen family. The specific biological roles of these proteins in virulence are not known.
The sequence of the alpha C-protein gene (bca) of GBS reveals four distinct domains: a signal sequence, an N-terminal region, a tandem repeat region, and a C-terminal anchor region (Michel, J. L., et al., Proc. Natl. Acad. Sci. USA. 89:10060-10064 (1992)). Presumably, the epsilon antigen gene (bce) has similar, though not necessarily identical domains. Identification and characterization of protective epitopes within the domains of the alpha, beta and epsilon C proteins will help determine the: immunological properties of these regions. These protective epitopes could be used to develop a C-protein-capsular polysaccharide conjugate vaccines to protect against a broad range of GBS strains.
The alpha antigen gene, bca, was previously cloned from the prototype Ia/C(xcex1/xcex2) strain A909. The antibodies raised to the cloned gene product were protective in an animal model (Michel, J. L. et al., Inf. Immun. 59:2023-2028 (1991)). In addition, the nucleotide sequence was determined and the derived animo acid sequence analyzed (Michel, J. L., et al., Proc. Natl. Acad. Sci. USA. 89:10060-10064 (1992)). The nucleotide sequence of alpha antigen revealed an open reading frame of 3,060 nucleotides encoding a precursor protein of 108.7 kilodaltons (kDa). The gene is composed of four distinct regions. Cleavage of a putative signal sequence of 56 amino acid yields a mature protein of 104.1 kDa. The 20.4 kDa N-terminal region shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein. The repeating units are identical, and each consists of 82 amino acids with a molecular mass of 8.7 kDa, which is encoded by 246 nucleotides. The C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of gram-positive surface-associated proteins (Michel, J. L., et al., Proc. Natl. Acad. Sci. USA 89(21):10060-10065 (1992)) including the group A streptococcal M proteins and the IgG-binding proteins of Staphylococcus (Fischetti, V., et al., Mol. Microbiol. 4:1603-1605, (1990); Wren, B. W., Mol Microbiol 5(4):797-803 (1991)). Immunoblot analysis of the native alpha antigen probed with either antisera to the cloned alpha antigen or an alpha antigen specific monoclonal antibody, 4G8, which binds to the repeat region, yields a regularly spaced ladder pattern of heterogeneous peptides. The bands are spaced at 8-kDa intervals, corresponding to the coding region defined by a single repeating subunit in the gene (Michel, J. L., et al., Proc. Natl. Acad. Sci. USA 89(21):10060-10065 (1992)). This correlation suggests that the repeat region is responsible for the laddered peptide heterogeneity of the alpha antigen.
It was reported that the size of the largest alpha antigen expressed by a given strain varies widely, from 54 to  greater than 200 kDa Opsonophagocytic killing in the presence of 4G8 antibodies correlated directly with increasing molecular mass of the alpha antigen and with the quantity of the alpha antigen expressed on the surface of GBS. GBS strains bearing the alpha antigen are resistant to killing by polymorphonuclear leukocytes in the absence of alpha antigen specific antibody. However, this resistance is not dependent on the overall size of the antigen expressed by a given strain. While a given strain produces an alpha antigen with a consistent size distribution, occasional colonies are isolated expressing smaller protein sizes; this phenomenon has also been observed in strain pairs from mothers and infants (Hervas, J. A. et al., Clin. Infect. Dis. 16:714-718 (1993)) In recent work, it was found that immune mice infected with GBS bearing the alpha antigen yielded strains with either smaller or absent alpha antigen expression (Beseth, B. D., A genetic analysis of phenotypic diversity of the C protein alpha antigen of group B Streptococcus, Bachelor of Arts thesis in Biology, Harvard College, (1992); Madoff, L. C., et al., Proc. Natl. Acad. Sci. USA. 93:4131-4136 (1996)).
To explore the molecular basis for the smaller size of alpha antigen seen in different strains of GBS, a panel of GBS isolates was examined and the size of the alpha antigen was compared with the size and composition of the alpha antigen gene. In doing so, it was discovered that the alpha antigen gene family was, in fact, composed of at least two different but related proteins. The new class of proteins has been named epsilon. The N-terminal region of the epsilon gene (bce), has been cloned and the nucleotide sequence analyzed. The molecular basis for size variation among alpha and epsilon bearing strains of GBS, and their potential for multiple intramolecular regions of antigenic variability, is potentially important both in understanding mechanisms of pathogenesis of GBS and in the development of a conjugate vaccine against GBS.
Development of an effective C-protein-based conjugate vaccine is assisted by a better understanding of the immunogenic and protection inducing effect of the alpha and epsilon antigens, particularly since the alpha antigen appears to undergo antigenic variation in isolates from neonates and their mothers (Hervas, J. A., et al., Clin. Infect. Dis. 16:714-718 (1993); Madoff, L. C., et al., Proc. Natl. Acad. Sci. USA. 93:4131-4136 (1996)). Deletions in the number of tandem repeats within the bca (Gravekamp, C., et al., Infect. Immun. 64:3576-3583 (1996) and the bce gene may give rise to antigenically variable polypeptides due to conformational epitopes that vary as a function of the number of repeats of the bce gene.
If an effective conjugate GBS vaccine is to be developed, protective epitopes that are conserved in the parental strains and their deletion mutants need to be identified. It has been observed that the bca gene was deleted in the neonatal isolates in the tandem repeat region but not in the N- and C-termini of the bca gene (Beseth, B. D., A genetic analysis of phenotypic diversity of the C protein alpha antigen of group B Streptococcus, Bachelor of Arts thesis in Biology, Harvard College, (1992); Madoff, L. C., et al., Proc. Natl. Acad. Sci. USA. 93:4131-4136 (1996)). Therefore, conserved epitopes are likely to be localized to the N- and C-terminal regions. The N-terminus of the alpha and epsilon C protein is a likely location for protective epitopes of these C-proteins that are conserved in spontaneous deletions and wild-type strains. However, the C-terminus of the alpha C protein may not contain protective epitopes, since it is thought to be involved in the antigen""s attachment to the cell-wall peptidoglycan (Michel, J. L., et al., Proc. Natl. Acad. Sci. USA. 89:10060-10064 (1992); Navarre, W. W. and Schneewind, O., Mol. Microbiol. 14:115-121 (1 994); Schneewind, O., et al., EMBO J. 12:4803-4811 (1993)).
The present invention concerns the development of a conjugate vaccine to group B Streptococcus (i.e. Streptococcus agalactiae) that utilizes the N-terminal region of the epsilon antigen or fragments thereof.
This novel conjugate vaccine should have the advantages both of eliciting T-cell dependent protection via the adjuvant action of the carrier protein and also providing additional protective epitopes that are present on the group B streptococcal protein (Insel, R. A, et al., New Eng. J. Med. (Editorial) 319(18):1219-1220 (1988); Baker, C. J, et al., Rev. of Infec. Dis. 7:458467 (1985)).
The advantage to use of the N-terminal region of the epsilon antigen in the vaccine is that the N-terminal is a likely location for protective epitopes of the epsilon antigen that are conserved in spontaneous deletions and wild-type strains. Further, the N-terminal region of the C-proteins may be more genetically stable than other regions of the molecule. The repeat regions of other C-proteins are known to contain antigenic epitotpes but also exhibit antigenic variability. (Klinge, et al., Infect. Immun. (April 1997, In press)).
In detail, the invention provides a conjugate vaccine capable of conferring host immunity to an infection by group B Streptococcus, the vaccine comprising (a) a polysaccharide conjugated to (b) a protein; wherein both the polysaccharide and the protein are characteristic molecules of the group B Streptococcus, and wherein the protein is a derivative of the C protein epsilon antigen N-terminal region that retains the ability to elicit protective antibodies against the group B Streptococcus.
The conjugate vaccine of the invention, can include the N-terminal region of the epsilon antigen or fragment thereof as the antigen in the vaccine plus the alpha and/or beta antigen or fragments thereof in combination with the epsilon antigen. These conjugate vaccines may also be used to obtain the passive vaccines of the invention.
The vaccine of the invention may also comprise the N-terminal region of the epsilon antigen or fragments thereof as the antigen in the vaccine either by itself or with the alpha and/or beta antigen or fragments thereof in combination with the epsilon antigen. Such fragments or combinations of fragments may be used in the vaccine of the invention in a non-conjugated form, i.e. not conjugated to a polysaccharide. These vaccines may also be used to obtain the passive vaccines of the invention.
The invention also concerns a method for preventing or attenuating an infection caused by a group B Streptococcus which comprises administering to an individual, suspected of being at risk for such an infection, an effective amount of the conjugate vaccine of the invention, such that it provides host immunity against the infection.
The invention further concerns a method for preventing or attenuating infection caused by a group B Streptococcus which comprises administering to a pregnant female an effective amount of a conjugate vaccine of the invention, such that it provides immunity to the infection to an unborn offspring of the female.
The invention also provides a method for preventing or attenuating an infection caused by a group B Streptococcus which comprises administering to an individual suspected of being at risk for such an infection an effective amount of an antisera elicited from the exposure of a second individual to a conjugate vaccine of the invention, such that it provides host immunity to the infection.
The invention also provides for the use of the N-terminal region of the epsilon antigen as an immunogenic composition and for use of such a composition in diagnostic procedures.
The invention also provides for a plasmid (ATCC Accession No. 98365, deposited Mar. 18, 1997 at The American, Type Culture Collection, Rockville, Md.) containing the N-terminal region of the epsilon antigen. The plasmid is referred to as pJMS36.